Significant progress in uncovering many of the molecular events with respect to control of bone formation has added considerably to our understanding of matrix mineralization. It is well established that insoluble collagen accumulation and cross-linking are essential for the development of a functional mineralized extracellular matrix. What is not known is how bone collagen maturation is regulated during development and whether cytokines modulate this process via the same mechanisms. Procollagen C-proteinase and lysyl oxidase have been identified as key players in the extracellular post-translational modification of collagen. In addition to processing procollagen, procollagen C-proteinase cleaves pro-lysyl oxidase into the 32 kDa active enzyme and the 18 kDa propeptide. Lysyl oxidase catalyzes the essential step necessary for collagen cross-linking. Recently, our laboratory has discovered a biological role for the 18 kDa propeptide in matrix mineralization. In Aim 1, the role of regulation of procollagen C-proteinase and lysyl oxidase in the formation of a mineralized extracellular matrix will be investigated in developing fetal rat calvaria osteoblastic cultures. Analyses will include a variety of molecular parameters including mRNA levels, protein levels, and enzyme activity, as well as assessment of extracellular matrix formation by measuring insoluble collagen and inorganic calcium accumulation. The recent development of knockout mice that lack the BPM-1 gene that encodes procollagen C-proteinase provides a supplementary approach to establish the role of procollagen C- proteinase and lysyl oxidase processing on in vitro bone formation. In Aim 2, the cytokine effects of TGF-beta and TNF-alpha on procollagen C- proteinase and lysyl oxidase, collagen synthesis, and collagen accumulation will be determined in developing osteoblastic cultures. In Aim 3, the osteogenic activity of the 18 kDa propeptide will be characterized. For this purpose recombinant lysyl oxidase propeptide will be prepared in a eukaryotic expression system and used to carry out functional studies and to explore receptor mediated mechanisms. The results obtained from these studies will add significantly to the understanding of how extracellular events lead to matrix maturation and mineralization. Once the molecular parameters addressed in this proposal are clearly defined, it may be possible to exploit the information gained for the development of therapeutic approaches in a variety of diseases affecting bone integrity.